Methods for isolating and preparing nucleic acids, both RNA and DNA, are used since several years in the state of the art and increasingly gain importance. Several purification methods are known, such as extraction/precipitation, chromatography, in particular adsorption chromatography, electrophoresis and affinity separation.
The known methods usually involve lysing the biological material by mechanical action and/or chemical action and/or biological action e.g. by treating the material with a detergent in the presence of protein degrading enzymes. Several different methods are also known for recovery of the nucleic acids after lysis. Some involve several extractions with organic solvents, for example with phenol and/or chloroform. These standard methods for isolating nucleic acids are very laborious and time-consuming. The relatively large number of steps required to purify nucleic acids from the starting materials also increase the risk of contaminations, especially with non-target nucleic acids.
Particularly useful are methods which are based on the non-sequence specific adsorption of the nucleic acids to the binding matrixes such as silica matrixes. Improved methods for isolating nucleic acids include the use of chaotropic salts, for example guanidinium chloride, and/or the use of alcohols, for example ethanol or isopropanol (see e.g. U.S. Pat. No. 5,234,809 and EP 1 146 049).
EP 0 389 063 also pertains to a process for isolating nucleic acids. The source containing the nucleic acids is lysed in the presence of chaotropic ions and then treated with a material which will adsorb nucleic acids, such as diatomaceous earth or other silica-containing mineral supports which are commonly used for nucleic acid isolations.
U.S. Pat. No. 5,155,018 discloses a process for isolation and purification of biologically active RNA from biological sources containing RNA, DNA and other cell contents. The source containing RNA is contacted with particles which consist of silica containing materials, such as finely dispersed glass. The binding buffer from which the RNA is adsorbed to the material comprises acidified solutions containing chaotropic salts. Under such conditions, RNA is specifically bound to the silica material.
WO 2005/045030 discloses an isolation and purification technology also incorporating the use of a porous matrix consisting of a material based on silica or of a silica coated material, which binds nucleic acids. However, the described method is performed in the absence of a chaotropic salts and alcohols.
The raw preparation of target nucleic acids is usually performed in order to enable subsequent analysis reactions. These subsequent reactions impose certain demands on both the isolation procedure and the purity and integrity of the isolated target nucleic acids. Especially when followed by enzymatic amplification reactions, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), NASBA (nucleic acid sequence-based amplification), or SSR (self-sustained sequence replication), the preparation of the target nucleic acids should be free of contaminants such as cell components and especially non-target nucleic acids in order to avoid false results.
Genomic DNA (gDNA) is a common contaminant of RNA isolations. Consequently, some commercially available RNA isolation kits provide a protocol for selective enzymatic removal of contaminating gDNA with deoxyribonuclease I (DNase I). However, treatment with DNase I occasionally results in a reduction of RNA yield and degradation of RNA by ribonucleases (RNases) that can contaminate commercially produced DNase I. Besides that, DNase I treatment adds hands-on time, extends the length of time required for the process, requires the addition of metal ions which can interfere with downstream processes and furthermore, it increases the overall costs of the RNA isolation.
Thus, it was attempted to improve the purity of the nucleic acid preparations, especially RNA preparations, wherein it was aimed to keep the content of contaminations such as genomic DNA as low as possible.
EP 0 818 461 e.g. discloses the use of lithium salts in the chaotropic lysis solution under certain conditions in order to increase the yield of pure RNA and in order to reduce genomic DNA contaminations. However, the obtained results are not satisfying.
EP 1 526 176 teaches the use of a pre-filtration technology in order to remove the genomic DNA from the RNA fraction. The method is based on the known RNA technology using chaotropic salts and a silica matrix for adsorbing the RNA. However, before the RNA is adsorbed at the silica matrix for isolation, the lysate is applied to a pre-filtration column in order to remove the DNA. During passage of the homogenate through the pre-filtration column, cellular contaminants, including genomic DNA, remain within the pre-filtration column while the effluent contains partially purified RNA. This RNA is then further purified by applying the effluent onto a second filtration column for adsorbing and washing the RNA. The drawback of this method is obvious, as two columns are necessary for purification which increases the hands-on time and costs.